Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury

ABSTRACT

This invention provides populations of neural cells bearing markers of glial cells, such as oligodendrocytes and their precursors. The populations are generated by differentiating pluripotent stem cells such as human embryonic stem cells under conditions that promote enrichment of cells with the desired phenotype or functional capability. Various combinations of differentiation factors and mitogens can be used to produce cell populations bearing markers of oligodendrocyte precursor cells. Upon further differentiation form complex processes characteristic of mature oligodendrocytes. The cells are capable of forming myelin sheaths, and can be used therapeutically improve function of the central nervous system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT application serial no.PCT/GB2003/003021, filed Jul. 10, 2003 (Atty Docket No. IRVN-009WO,entitled “Oligodendrocytes Derived from Human Embryonic Stem Cells froRemyelination and Treatment of Spinal Cord Injury,” which applicationwas filed in English and designating the United States, whichapplication is a continuation-in-part of U.S. application Ser. No.10/406,817, filed Apr. 4, 2003, which application claims the benefit ofU.S. provisional application Ser. No. 60/395,382, filed Jul. 11, 2002.The disclosures of each of these applications is incorporated herein byreference in their entireties.

TECHNICAL FIELD

This invention relates generally to the field of cell biology ofembryonic cells and neural progenitor cells. More specifically, thisinvention provides enriched populations of oligodendrocytes and theirprecursors, suitable for use in biological research, drug screening, andhuman therapy.

BACKGROUND

Oligodendrocytes play a vital physiological role in support of thecentral nervous system. Availability of oligodendrocytes for humantherapy may facilitate healing of disabling conditions that result fromdefects in the myelin sheath that insulates nerve cells.

Multiple Sclerosis is a progressive and disabling demyelination disease,involving gradual destruction of the myelin sheath that surrounds thenerve cells in the brain and spinal cord. Symptoms range from numbness,visual impairment and cognitive changes, to paralysis. The disease isbelieved to have immunological and genetic components, often appearingin clinical form between ages 20 and 40. It affects about 300,000 peoplein the U.S. alone. Therapeutic modalities currently involve courses ofβ-interferon or corticosteroids. These drugs may shorten the symptomaticperiod during attacks, but generally do not prevent long-termdisability.

Traumatic injury to the spinal cord causes demyelination of intact axonsnear the trauma site, which robs them of their capacity for neuraltransmission. There are about 11,000 new cases of spinal cord injuryevery year in the U.S. The SCI Information Network estimates that thelifetime direct costs to patients suffering from incomplete motorfunction at any level ranges from $400,000 to $2,200,000, not includinglost wages and effects on the quality of life.

Myelin on cells of the central nervous system is put in place byoligodendrocytes, which wrap around axons in the vicinity, forming amyelin sheath. The role of oligodendrocytes and their progenitors indisease conditions is reviewed by Keirstead & Blakemore (Adv. Exp. Med.Biol. 468:183, 1999). Oligodendrocyte progenitors known as O-2A cellsare present in normal adult CNS and in lesions of Multiple Sclerosis,and participate in remyelination (Scolding et al., Brain 121:2221, 1998;and Scolding et al., Neuroscience 89:1, 1999). Failure to remyelinateadequately may occur because oligodendrocytes proliferate symmetrically,using up the reservoir of progenitor cells where the damage isextensive.

Considerable research work has been done with a view to creating cellpopulations that could be used in regenerative medicine to restoreneurological function (reviewed by Park et al., J. Neurotrauma 16:675,1999). Keirstead et al. (J. Neuroscience 19:7529, 1999) isolated CNSprecursors from the postnatal rat brain that generate oligodendrocytesand Schwann cells after transplantation. Svendsen et al. (J. Neurosci.Meth. 85:141, 1998) isolated precursor cells from the developing humancortex. Mujtaba et al. (Dev. Biol. 214:113, 1999) report isolation ofneural precursors from embryonic stem cells.

PCT publication WO 97/07200 (Stanford U.) shows cultures ofoligodendrocyte precursors isolated from adult rat brain. PCTpublication WO 01/28342 (Washington U.) proposes certain methods forculturing nerve cells in preconditioned oligodendrocyte culture medium.U.S. Pat. No. 5,753,506 (Johe, CNS Stem Cell Technology) relates to aculture system for maintaining stem cells isolated from neural tissuethat have the capacity to differentiate into neurons, astrocytes, andoligodendrocytes. U.S. Pat. No. 6,238,922 (StemCells Inc.) proposesdissociation of neural tissue into cells that have the capability ofdifferentiating into neurons and glia. U.S. Pat. No. 6,235,527 (Rao etal., U. Utah) relates to populations of mammalian CNS glial-restrictedprecursor cells dissociated from mammalian neural tube tissue andselected based on the A2B5 cell surface marker.

U.S. Pat. No. 5,968,829 (Cytotherapeutics) claims culture mediumcontaining CNS neural stem cells that have the capacity to produceneurons, astrocytes, and oligodendrocytes. PCT publication WO 97/32608pertains to genetically engineered primary oligodendrocytes frotransplantation-mediated delivery in the CNS. U.S. Pat. No. 5,830,621(Signal Pharmaceuticals) describes a human oligodendrocyte cell linedeposited with the ATCC under Accession No. CRL 11881. The line isessentially free of the characteristic markers GFAP, GalC, O4, and A2B5.

Unfortunately, it is not yet clear whether progenitors isolated fromneural tissue will have sufficient replicative capacity to produce thenumber of cells necessary for human clinical therapy.

An alternative source is pluripotent cells isolated from early embryonictissue. Embryonic stem (ES) cells were first isolated from mouse embryosover 25 years ago (G. R. Martin, Proc. Natl. Acad. Sci. U.S.A. 78:7634,1981). ES cells are believed to be capable of giving rise to progeny ofvirtually any tissue type of the same species.

Fraichard et al. (J. Cell Sci. 108:3181, 1995) report in vitrodifferentiation of mouse ES cells into glial cells and functionalneurons. Mujtaba et al. (Dev. Biol. 214:113, 1999) report isolation ofneural precursors from mouse ES cells. Li, Smith et al. (Cur. Biol.8:971, 1998) report generation of neuronal precursors from mouse EScells by lineage selection. Brüstle, McKay et al. (Proc. Natl. Acad.Sci. USA 94:14809, 1997; Science 285:754, 1999) report glial precursorsderived from mouse ES cells as a potential source of myelinatingtransplants. McDonald et al. (Nat. Med 5:1410, 1999; Proc. Natl. Acad.Sci. USA 97:6126, 2000) report that mouse ES cells form oligodendrocytesand myelinate in culture and after spinal cord transplantation.

Human ES cells were not isolated until much more recently (Thomson etal., Science 282:114, 1998). Human ES cells require very differentconditions to keep them in an undifferentiated state, or direct themalong particular differentiation pathways (U.S. Pat. Nos. 6,090,622 &6,200,806; PCT publications WO 99/20741 & WO 01/51616). For this reason,much less is known about how to prepare relatively homogeneouspopulations of differentiated cells from human ES cells.

PCT publication WO 01/88104 (Carpenter, Geron Corporation) describesneural progenitor cell populations obtained by differentiating human EScells. Populations have been obtained that are over 90% NCAM positive,35% β-tubulin positive, and 75% A2B5 positive. Zhang et al. (NatureBiotech. 19:1129, 2001) report differentiation of transplantable neuralprecursors from human ES cells. International Patent ApplicationPCT/US02/19477 (Carpenter et al., Geron Corporation) describes ES cellderived neural cell populations in which at least 10% of the MAP-2positive cells in the produced population express tyrosine hydroxylase,a marker for dopaminergic neurons.

Recently, Billon et al. (J. Cell Sci. 115:3657, 2002) describe thetiming of oligodendrocyte development from genetically engineered mouseES cells. Kuo et al. (Biol. Reprod. Dec. 11, 2002) report a populationof monkey ES derived cells that were 28% GFAP positive; and Xian et al.(Stem Cells 21:41, 2003) report generation of oligodendrocytes frommouse ES cells using the lineage-specific transcription factor Olig2.

In order to realize the full potential of pPS cells in the management ofhuman health and disease, it is necessary to develop new paradigms togenerate enriched populations of cells useful for treating demyelinationconditions.

SUMMARY

This invention provides a system for efficient production of primatecells of the glial lineage for use in research or the preparation ofpharmaceutical compositions.

The differentiated cell populations of this invention are isolated orcultured in vitro, and are highly enriched for characteristics of glialcells, or cells capable of myelinating neural tissue. The cells can havemorphological features of oligodendrocytes, expression of certainantibody-detectable or amplifiable markers listed later in thisdisclosure, or the ability to form oligodendrocytes upon furtherdifferentiation. The cells can also have certain functional features ofoligodendrocytes, such as the ability to myelinate ganglia in acoculture assay, the ability to restore myelin to demyelinated axons invivo, or the ability to improve neurological function in humans ornon-human animals. One, two, three, or more of these features may bepresent in any combination.

The cell populations can be prepared from less differentiated stem cellsof various kinds. Potential originating cells include primatepluripotent stem (pPS) cells derived from blastocysts (exemplified byembryonic stem cells) or the germinal tissue of early embryos.Accordingly, the cells will have the characteristic of being the progenyof the tissue from which they originated, which can be confirmed byshowing that the originating cells and the differentiated cells have thesame genome.

Other aspects of the invention relate to methods for generating ormaintaining the differentiated cells already described. The methodsinvolve culturing multipotential or pluripotential stem cells in thepresence of one or more growth or differentiation factors, such as thoseillustrated later in the disclosure.

By way of example, stem cells can be cultured in a medium containing oneor more differentiation factors such as triiodothyronine (T3), selenium,or retinoic acid, with or without a mitogen such as fibroblast growthfactor (FGF). Initial formation of the differentiated cells can occur insuspension culture, where oligodendrocyte lineage cells may formrelatively homogeneous spheres. Other cell types can be removed by asuitable separation means, such as plating the culture onto a surfacethat selectively adheres the desired cell type. Optionally, thedifferentiated cells can be caused to proliferate further before orafter selection by culturing with a mitogen such as FGF or epidermalgrowth factor, often in the presence of one or more differentiationfactors, such as those used in the original derivation of the cells.Subsequently, the cells can optionally be caused to maturate further byculturing in the absence of mitogens, or in the presence of a surfacesuch as poly-L-lysine that enhances late-stage differentiation.

The cells of this invention can be used for a number of commerciallyimportant applications. For example, the cells are suitable forscreening compounds for an effect on glial cells, in which the presenceof the compound is correlated with cell maintenance, toxicity, furtherdifferentiation, or the ability to function as a glial cell. The cellsare also suitable for causing myelination of neighboring neurologicaltissue, both in vitro and in vivo.

The cells of this invention can also be used in the preparation ofpharmaceutical compositions for human or animal therapy. Thecompositions can then be used to treat conditions such as thoseassociated with a defect in the myelination of axons, or spinal cordinjury.

Further aspects of the invention will be apparent from the descriptionthat follows.

DRAWINGS

FIG. 1 is a phase contrast image taken using an inverted microscope,showing human embryonic stem (ES) cells that have been cultured 2 daysin suspension in a medium containing basic FGF, and oligodendrocytedifferentiation factors.

FIG. 2 shows the cells after culturing for 7 days with retinoic acid.Large clear spheres of cells were present, representing 80-90% of thecells in the culture.

FIG. 3 shows what happens after retinoic acid is removed, and the cellsare maintained in the presence of a low concentration of FGF.

FIG. 4 shows the changes appearing after FGF was removed. The largeaggregates dissociated, and the entire culture medium was flooded withsingle cells and small clusters. At the same time, new bright yellowspheres were observed (arrows).

FIG. 5 shows bright-yellow spheres (arrows) growing as cells werecultured with epidermal growth factor, in the absence of FGF.

FIG. 6 shows migration and branching of the glial committed neuralprecursors from the spheres 2 or 3 days after plating on Matrigel®.

FIG. 7 shows the results in which oligodendrocyte lineage cells wereselected by adherence onto Matrigel® for just 10-20 hours. Non-adherentcells were discarded, which eliminates virtually all of the cells notbearing oligodendrocyte markers. The adherent cells were thenresuspended and expanded for 7 days in the presence of FGF, EGF, andglial precursor medium. This has the advantage of producing a moredisperse cell population more suited for therapeutic administration andother purposes. The cells were then matured on poly-L-lysine laminin inthe absence of mitogens. This micrograph shows staining forgalactocerebroside (GalC; open arrows), with cell nuclei counterstainedusing hematoxin (closed arrows). The percentage of cells staining forGalC under these conditions was at least ˜95%.

FIG. 8 shows a higher magnification of ES-derived oligodendrocytes. Thecells have morphological features that are characteristic of matureoligodendrocytes: numerous complex processes that appear to have myelinwebbing in between.

FIG. 9 shows the progression of cell morphology during differentiation.(A): Undifferentiated hES cells. (B): Transparent spheres growing fromembryoid bodies in suspension cultures containing retinoic acid. (C):Expansion of yellow spheres containing oligodendrocyte precursors cellsin the presence of EGF. (D) Oligodendrocyte lineage cells positivelyselected by plating onto Matrigel®. (E, F): Increasing prominence ofoligodendrocyte precursors in the culture. (G, H): Subsequent platingcauses further differentiation to mature oligodendrocytes.

FIG. 10 shows an immunocytochemical analysis of the matureoligodendrocytes. One week after plating, the cells are positive for theearly glial cell marker NG2 (A). Eight weeks after plating, the cellsare positive for GalC (C), O4 (D), and RIP (E). Panel (B) shows numberof cells staining with indicated markers.

FIG. 11 shows tissue sections of oligodendrocyte precursors administeredto the spinal cord of rats, stained with antibody specific for humannuclear protein.

FIG. 12 shows that nine weeks after engraftment, the cells have migratedor proliferated into the white matter.

FIG. 13 shows the engrafted oligodendrocytes do not worsen the injurycaused by secondary enlargement following contusion injury, measured asthe cross-sectional area of the cord.

FIG. 14 shows that hES cells induce axonal branching. New axons areshown in the images from the oligodendrocyte engrafted animals (upperpanels) as dark narrow lines of BDA staining. No branching was observedin untreated animals. The engrafted cells are inducing regenerativeplasticity.

FIG. 15 shows quantitation of the neuronal branching from the site ofinjury (the epicenter) (mean±SEM for 3 sections per block). Treatedanimals had labeled axons at a significantly higher level right up tothe epicenter on the rostral side of the injury.

FIG. 16 shows evidence of substantial remyelination in the animalsengrafted with hES derived oligodendrocytes. The thick circle in theupper electron micrograph is a normally myelinated fiber. The rest ofthe axons in the field show a thin layer of myelin. The axon in theupper right panel shows about 5 or 6 wraps, and evidence of ongoingremyelination. Only engrafted animals showed new myelin attributable tooligodendrocyte activity. This provides a mechanism to explain thebehavioral improvement in the engrafted animals.

FIG. 17 shows markers detected on undifferentiated hES cell colonies(Left side: antibody plus DAPI; right side: antibody staining alone).Top row shows colonies labeled for SSEA-4, a marker of pluripotentcells. Bottom row shows stromal cells surrounding the colonies labeledfor the mesodermal marker BMP4.

FIG. 18 shows the transient appearance of transcription factor Pax6during differentiation. Top row shows staining towards the center of thecluster on day 10, already regulated down in the more differentiatedcells towards the periphery. Bottom row shows virtually no staining atday 35 of differentiation.

FIG. 19 shows markers detected in early-stage oligodendrocyte lineagecells, present at day 10 of differentiation. Top row: the transcriptionfactor Olig1. Middle row: the transcription factor SOX10. Bottom row:oligodendrocyte progenitor marker A2B5.

FIGS. 20A-20B show markers predominating in the oligoprogenitorssuitable for transplant at day 35. First and second rows (20A): NG2(chondroitin sulfate proteoglycan, a marker of oligodendrocyteprecursors); Third row (20A): GalC; Fourth row (20B): O4; Fifth row(20B): Tuj1 (a marker of neurons). Virtually all of the cells bearoligodendroglial markers, but not markers for neuronal cells,mesenchymal cells, or undifferentiated hES cells (20B, bottom graph).

FIG. 21 shows the results of oligodendrocytes administered to Shiverermice, which are genetically deficient in the ability to make myelinbasic protein. Ultrastructurally, axons of shiverer mice are devoid ofmyelin or are surrounded by one or two uncompacted wraps of myelin(Upper Panel). Six weeks after transplantation of cells, electronmicroscopic analysis indicated multilayered compact myelin indicting themyelinogenic capability of the transplant population (Lower Panel). Thecompact myelin is being produced directly by the administeredoligodendrocytes.

FIG. 22 shows the results of an experiment in which rats were contusedin the spinal cord using an impactor, and then evaluated for spinal cordfunction in the ensuing period by quantitation on the BBB scale. Uppergraph: 200 kiloDyne contusion; lower graph: 250 kiloDyne contusion. (▪)animals treated with ES-derived oligodendrocytes 1 week after injury(n=5); (▴) control animals receiving the same contusion but noadministered cells (n=3). Mean±SEM, for assessments done blinded.Animals engrafted with hES derived oligodendrocytes showed significantlybetter overground locomotion, persisting for more than 5 weeks aftertreatment.

DETAILED DESCRIPTION

This invention solves the problem of generating large populations ofoligodendrocytes and their precursors by producing them efficiently frommultipotent stem cells.

The differentiated cell populations are remarkably homogeneous. FIG. 7shows an exemplary preparation of human cells with morphologicalfeatures characteristic of oligodendrocytes. The population has beenstained for GalC, a marker for cells of the oligodendrocyte lineage.FIG. 8 is a higher magnification view, showing the production of sheetsof myelin poised to myelinate any neurons in the vicinity. Thefunctional properties of these cells make them well suited for furthercharacterization of the properties of oligodendrocytes, and for use inhuman therapy.

An ideal source of the cells of this invention is primate pluripotentstem (pPS) cells of various kinds. pPS cells can be coaxed into theoligodendrocyte pathway by selecting from amongst several suitableculture conditions and cofactors according to the strategy describedbelow. It has been discovered that conversion of undifferentiated pPScells into oligodendrocytes can be done at an efficiency of as much as25% under optimized conditions.

The compositions and methods of this invention provide importantadvantages over previously available technology. Oligodendrocytes can begenerated having any histocompatibility type, by differentiating themfrom pPS cells of the desired allotype. If desired, the cells can begenetically modified before or after differentiation in any way thatenhances engraftment. Since the cells are derived from a natural cellline but do not require ongoing tissue dissection, they are ideal formeeting the quality control requirements for regulatory approval.

Importantly, there is an almost limitless supply of cells that can beproduced from each starting stem cell population. As illustrated inExample 1, once oligodendrocyte lineage cells have been generated andselected, they can be expanded considerably by culturing in the presenceof growth factors. Furthermore, the originating pPS cells can be causedto proliferate almost indefinitely, thereby providing a continual sourcefor more differentiated cells.

The disclosure that follows provides further description of how to makeand use the differentiated cells of this invention. The populations areremarkably uniform, and therefore suitable for use in a number ofcommercially important applications.

One aspect of the invention provides a differentiated cell population,in which at least about 80% of cells are progeny of primate pluripotentstem (pPS) cells; stain with antibody specific for NG2proteoglycan (orother oligodendrocyte markers); and are negative for NeuN (or othermarkers of neuronal cells and other potential contaminants). Thedifferentiated cell population may be part of a system of componentssuitable for generating oligodendrocyte lineage cells. The system mayfurther comprise the line of pPS cells (such as human embryonic stemcells) from which the differentiated cells were produced. Optionally, atleast 80% of the cells of the differentiated cell population alsoexpress A2B5 or PDGFRα. At least 20% of the cells may show a bipolarmorphology characteristic of oligodendrocyte precursors.

Under some circumstances, the differentiated cell populations causesdeposition of compact myelin around neuronal axons followingimplantation of the population into the spinal cord of a shiverer mutantmouse; or causes improvement in overground locomotion followingimplantation of the population in or around the spinal cord in acontusion-injured rat. Alternatively, the oligodendrocyte precursor cellpopulations of this invention may be further differentiated in vitro(for example, by culturing for 3 days on poly-L-lysine and laminin inthe absence of mitogens). This may generate a more mature population inwhich at least 10% of the cells have complex processes characteristic ofmature oligodendrocytes; and about 80%, 90%, 95% or more have matureoligodendrocyte markers such as GalC. The oligodendrocyte lineage cellsmade during the proliferative phase can be characterized not only by themarkers they are expressing in the progenitor phase, but also theircapacity to generate enriched populations of mature cells or theircapacity to perform desirable functions in vivo.

As illustrated below, some differentiated cell populations of thisinvention can be obtained by a process in which the undifferentiated pPScells are cultured in a medium containing a mitogen and at least twooligodendrocyte differentiation factors. For example, theundifferentiated pPS cells may be cultured in suspension so as to formcell aggregates in the presence of basic fibroblast growth factor (FGF),triiodothyronine (T3), retinoic acid, and perhaps selenium.

Further explanation and illustration of the invention is provided in thesections that follow.

DEFINITIONS

Oligodendrocytes are neural cells of ectodermal origin, forming part ofthe adventitial structure (neuroglia) of the central nervous system.They have variable numbers of veil-like or sheet-like processes thatwrap around individual axons to form the myelin sheath of the CNS. Theycan be identified by morphological, phenotypic, or functional criteriaas explained later in this disclosure.

“Neural precursor cells” or “neuroepithelial stem cells” are cells thatcan generate progeny that are either neuronal cells (such as neuronalprecursors or mature neurons) or glial cells. The term “glial cells”encompasses mature oligodendrocytes, astrocytes, and committedprecursors for either or both of these cell types.

“Oligodendrocyte precursors” are neural cells that are committed to formprogeny comprising mature oligodendrocytes and/or more precursor cells,in preference to neurons or non-neurological tissue. Unless otherwisespecified, they may but do not necessarily have the capability of makingother types of glial cells, such as astrocytes. Reference in thisdisclosure to “oligodendrocytes” or “oligodendrocyte lineage cells”refers to both oligodendrocyte precursors and mature cells, unlessotherwise indicated.

In the context of cell ontogeny, the adjective “differentiated” is arelative term. A “differentiated cell” is a cell that has progressedfurther down the developmental pathway than the cell it is beingcompared with. Thus, pluripotent embryonic stem cells can differentiateto lineage-restricted precursor cells, such as the various types ofprecursors listed above. These in turn can be differentiated further tocells further down the pathway, or to an end-stage differentiated cell,such as mature oligodendrocytes.

A “differentiation agent”, as used in this disclosure, refers to one ofa collection of compounds that are used in culture systems of thisinvention to produce differentiated cells of the oligodendrocyte lineage(including precursor cells and terminally differentiated cells). Nolimitation is intended as to the mode of action of the compound. Forexample, the agent may assist the differentiation process by inducing orassisting a change in phenotype, promoting growth of cells with aparticular phenotype or retarding the growth of others, or acting inconcert with other agents through unknown mechanisms.

Unless otherwise specified, reference to “selenium” in this disclosurerefers to any oxidation form of Se, including selenite (SeO32-),selenate (SeO42-), or senenide (Se2-) in solution with any counter-ion.

Prototype “primate Pluripotent Stem cells” (pPS cells) are pluripotentcells derived from pre-embryonic, embryonic, or fetal tissue at any timeafter fertilization, and have the characteristic of being capable underappropriate conditions of producing progeny of several different celltypes that are derivatives of all of the three germinal layers(endoderm, mesoderm, and ectoderm), according to a standard art-acceptedtest, such as the ability to form a teratoma in 8-12 week old SCID mice.Included in the definition of pPS cells are embryonic cells of varioustypes, exemplified by human embryonic stem (hES) cells, and humanembryonic germ (hEG) cells. The pPS cells are preferably not derivedfrom a malignant source. It is desirable (but not always necessary) thatthe cells be euploid. Depending on their source and method of culture,the pPS cells may or may not be totipotent, in the sense that they havethe capacity of developing into all the different cell types of thehuman body.

pPS cell cultures are described as “undifferentiated” when a substantialproportion of stem cells and their derivatives in the population displaymorphological characteristics of undifferentiated cells, distinguishingthem from differentiated cells of embryo or adult origin. It isunderstood that colonies of undifferentiated cells within the populationwill often be surrounded by neighboring cells that are differentiated.

“Feeder cells” or “feeders” are terms used to describe cells of one typethat are co-cultured with cells of another type, to provide anenvironment in which the cells of the second type can grow. pPS cellpopulations are said to be “essentially free” of feeder cells if thecells have been grown through at least one round after splitting inwhich fresh feeder cells are not added to support the growth of pPScells.

The term “embryoid bodies” refers to aggregates of differentiated andundifferentiated cells that appear when pPS cells overgrow in monolayercultures, or are maintained in suspension cultures. Embryoid bodies area mixture of different cell types, typically from several germ layers,distinguishable by morphological criteria and cell markers detectable byimmunocytochemistry.

A “growth environment” is an environment in which cells of interest willproliferate, differentiate, or mature in vitro. Features of theenvironment include the medium in which the cells are cultured, anygrowth factors or differentiation-inducing factors that may be present,and a supporting structure (such as a substrate on a solid surface) ifpresent.

In assessment of phenotypic markers on individual cells or cellpopulations, unless stated wise, the cell is said to be “positive” forthe marker if it shows substantially higher staining using specificantibody in a secondary immunocytochemistry stain, compared with isotypecontrol. Unless stated otherwise, the cell is said to be “negative” ifthe marker is not antibody detectable by this type of immunocytochemicalanalysis.

A cell is said to be “genetically altered”, “transfected”, or“genetically transformed” when a polynucleotide has been transferredinto the cell by any suitable means of artificial manipulation, or wherethe cell is a progeny of the originally altered cell that has inheritedthe polynucleotide. The polynucleotide will often comprise atranscribable sequence encoding a protein of interest, which enables thecell to express the protein at an elevated level. The genetic alterationis said to be “inheritable” if progeny of the altered cell have the samealteration.

General Techniques

General methods in molecular genetics and genetic engineering aredescribed in the current editions of Molecular Cloning: A LaboratoryManual, (Sambrook et al., Cold Spring Harbor); Gene Transfer Vectors forMammalian Cells (Miller & Calos eds.); and Current Protocols inMolecular Biology (F. M. Ausubel et al. eds., Wiley & Sons). Cellbiology, protein chemistry, and antibody techniques can be found inCurrent Protocols in Protein Science (J. E. Colligan et al. eds., Wiley& Sons); Current Protocols in Cell Biology (J. S. Bonifacino et al.,Wiley & Sons) and Current protocols in Immunology (J. E. Colligan et al.eds., Wiley & Sons.). Reagents, cloning vectors, and kits for geneticmanipulation referred to in this disclosure are available fromcommercial vendors such as BioRad, Stratagene, Invitrogen, ClonTech, andSigma-Aldrich Co.

Cell culture methods are described generally in the current edition ofCulture of Animal Cells: A Manual of Basic Technique (R. I. Freshneyed., Wiley & Sons); General Techniques of Cell Culture (M. A. Harrison &I. F. Rae, Cambridge Univ. Press), and Embryonic Stem Cells: Methods andProtocols (K. Turksen ed., Humana Press). Tissue culture supplies andreagents are available from commercial vendors such as Gibco/BRL,Nalgene-Nunc International, Sigma Chemical Co., and ICN Biomedicals.

Specialized reference books relevant to this disclosure includePrinciples of Neuroscience, 4^(th) Edition, Kandel et al. eds.,McGraw-Hill 2000; CNS Regeneration: Basic Science and Clinical Advances,M. H. Tuszynski & J. H. Kordower, eds., Academic Press, 1999; TheNeuron: Cell and Molecular Biology, 3^(rd) Edition, I. B. Levitan & L.K. Kaczmarek, Oxford U. Press, 2001; Glial Cells: Their Role inBehaviour, P. R. Laming et al. eds., Cambridge U. Press, 1998; TheFunctional Roles of Glial Cells in Health and Disease, Matsas &Tsacopoulos eds., Plenum Pub. Corp, 1999; Glial Cell Development, Jessen& Richardson eds., Oxford U. Press, 2001; and Man of Steel, AdrianHavill, 1996.

Sources of Stem Cells

This invention can be practiced using stem cells of various types.Particularly suitable for use in this invention are primate pluripotentstem (pPS) cells derived from tissue formed after gestation, such as ablastocyst, or fetal or embryonic tissue taken any time duringgestation. Non-limiting examples are primary cultures or establishedlines of embryonic stem cells or embryonic germ cells, as describedbelow. The techniques of this invention can also be implemented directlywith primary embryonic or fetal tissue, deriving neural cells directlyfrom primary embryonic cells without first establishing anundifferentiated cell line.

The illustrations provided in the Example section ensue from work donewith human embryonic stem cells. However, except where otherwisespecified, the invention can be practiced using stem cells of anyvertebrate species. Included are pluripotent stem cells from humans; aswell as non-human primates, and other non-human mammals.

Embryonic Stem Cells

Embryonic stem cells can be isolated from primate tissue (U.S. Pat. No.5,843,780; Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995).Human embryonic stem (hES) cells can be prepared from human blastomeresusing techniques described by Thomson et al. (U.S. Pat. No. 6,200,806;Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) andReubinoff et al, Nature Biotech. 18:399, 2000. Equivalent cell types tohES cells include their pluripotent derivatives, such as primitiveectoderm-like (EPL) cells, as outlined in WO 01/51610 (Bresagen).

In one method, the zona pellucida is removed from developed blastocystsby brief exposure to pronase (Sigma). The inner cell masses are isolatedby immunosurgery, in which blastocysts are exposed to a 1:50 dilution ofrabbit anti-human spleen cell antiserum for 30 min, then washed for 5min three times in DMEM, and exposed to a 1:5 dilution of Guinea pigcomplement (Gibco) for 3 min (Solter et al., Proc. Natl. Acad. Sci. USA72:5099, 1975). After two further washes in DMEM, lysed trophectodermcells are removed from the intact inner cell mass (ICM) by gentlepipetting, and the ICM plated on mEF feeder layers.

After 9 to 15 days, inner cell mass-derived outgrowths are dissociatedinto clumps, either by exposure to calcium and magnesium-freephosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispaseor trypsin, or by mechanical dissociation with a micropipette; and thenreplated on mEF in fresh medium. Growing colonies havingundifferentiated morphology are individually selected by micropipette,mechanically dissociated into clumps, and replated. ES-like morphologyis characterized as compact colonies with apparently high nucleus tocytoplasm ratio and prominent nucleoli. Resulting ES cells are thenroutinely split every 1-2 weeks by brief trypsinization, exposure toDulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase(˜200 U/mL; Gibco) or by selection of individual colonies bymicropipette. Clump sizes of about 50 to 100 cells are optimal.

Embryonic Germ Cells

Human Embryonic Germ (hEG) cells can be prepared from primordial germcells as described in Shamblott et al., Proc. Natl. Acad. Sci. USA95:13726, 1998 and U.S. Pat. No. 6,090,622.

Briefly, genital ridges taken after ˜8-11 weeks are rinsed with isotonicbuffer, then placed into 0.1 mL 0.05% trypsin/0.53 mM sodium EDTAsolution (BRL) and cut into <1 mm³ chunks. After disaggregation, thecells are incubated 1 h or overnight at 37° C. in ˜3.5 mL EG growthmedium (DMEM containing D-glucose, NaHCO₃; 15% ES qualified fetal calfserum; 2 mM glutamine; 1 mM sodium pyruvate; 1000-2000 U/mL humanrecombinant leukemia inhibitory factor; 1-2 ng/ml human recombinantbFGF; and 10 μM forskolin (in 10% DMSO).

The cells are then resuspended in 1-3 mL of EG growth medium, and platedonto a feeder layer (e.g., STO cells, ATCC No. CRL 1503, inactivatedwith 5000 rad γ-irradiation). The first passage is done after 7-10 days,and then cultured with daily replacement of medium until cell morphologyconsistent with EG cells is observed, typically after 7-30 days or 1-4passages.

Other Stem Cells

By no means does the practice of this invention require that a humanembryo or blastocyst be disaggregated in order to produce the pPS orembryonic stem cells useful as the starting material for makingoligodendrocytes. hES cells can be obtained from established linesobtainable from public depositories (for example, the WiCell ResearchInstitute, Madison Wis. U.S.A., or the American Type Culture Collection,Manassas Va., U.S.A.). U.S. Patent Publication 2003-0113910 A1 reportspluripotent stem cells derived without the use of embryos or fetaltissue. It may also be possible to reprogram cord blood or otherprogenitor cells into pPS cells by using a factor that induces thepluripotent phenotype (Chambers et al., Cell 113:643, 2003; Mitsui etal., Cell 113:631, 2003). Under appropriate conditions, any cell thatotherwise meets the definitions for pPS or hES cells can be used for thederivation of oligodendrocyte lineage cells according to this invention.

Some of the techniques provided in this disclosure can also be used tomaintain or advance the differentiation of more committed cell types,such as ectoderm cells, and neural cells or neural precursors obtainedfrom fetal or adult tissue. Methods of obtaining such cells aredescribed, for example, in U.S. Pat. Nos. 5,852,832; 5,654,183;5,849,553; and 5,968,829; and PCT publications WO 98/50526 and WO99/01159.

Propagation of pPS Cells in an Undifferentiated State

pPS cells can be propagated continuously in culture, using cultureconditions that promote proliferation without promoting differentiation.Exemplary serum-containing ES medium is made with 80% DMEM (such asKnockout DMEM, Gibco), 20% of either defined fetal bovine serum (FBS,Hyclone) or serum replacement (WO 98/30679), 1% non-essential aminoacids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Just before use,human bFGF is added to 4 ng/mL (WO 99/20741, Geron Corp.).

The pPS cells can be expanded in the undifferentiated state only byculturing in an environment that inhibits differentiation.Traditionally, pPS cells are cultured on a layer of feeder cells derivedfrom embryonic or fetal tissue of the mouse. Culture plates are platedwith 375,000 irradiated mEFs per well, irradiated to inhibitproliferation but permit synthesis of factors that support pPS cells,and used 5 h to 4 days after plating (U.S. Pat. No. 6,200,806). Humanfeeder cells have recently been developed that support proliferation ofhuman embryonic stem cells without differentiation (WO 01/51616; U.S.Ser. No. 09/888,309; Geron Corp.). The cells are obtained bydifferentiating hES cells, selecting cells that have the desiredactivity, and then immortalizing them by transfecting them to expresstelomerase reverse transcriptase.

pPS cells can be maintained in an undifferentiated state even withoutfeeder cells. The environment for feeder-free cultures includes asuitable culture substrate, particularly an extracellular matrix such asMatrigel® or laminin. The pPS cells are plated at >15,000 cells cm⁻²(optimally 90,000 cm⁻² to 170,000 cm⁻²). Feeder-free cultures aresupported by a nutrient medium containing factors that supportproliferation of the cells without differentiation. Such factors may beintroduced into the medium by culturing the medium with cells secretingsuch factors, such as irradiated (˜4,000 rad) primary mouse embryonicfibroblasts, telomerized mouse fibroblasts, or human feeder cellsderived from pPS cells. Medium can be conditioned by plating the feedersat a density of ˜5-6×10⁴ cm⁻² in a serum free medium such as KO DMEMsupplemented with 20% serum replacement and 4 to 8 ng/mL bFGF. Mediumthat has been conditioned for 1-2 days is supplemented with furtherbFGF, and used to support pPS cell culture for 1-2 days. Features of thefeeder-free culture method are further discussed in International PatentPublications WO 99/20741 & WO 01/51616; and Xu et al., Nat. Biotechnol.19:971, 2001.

Under the microscope, ES cells appear with high nuclear/cytoplasmicratios, prominent nucleoli, and compact colony formation with celljunctions that are difficult to discern. Primate ES cells typicallyexpress stage-specific embryonic antigens (SSEA) 3 and 4, markersdetectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson etal., Science 282:1145, 1998), and telomerase activity. Differentiationof pPS cells in vitro results in the loss of SSEA-4, Tra-1-60, andTra-1-81 expression, and increased expression of SSEA-1, which is alsofound on undifferentiated hEG cells.

Making Oligodendrocytes from Stem Cells

Oligodendrocyte lineage cells of this invention are obtained byculturing stem cells in a special growth environment that enriches andexpands cells with the desired phenotype. The growth environment mayspecifically direct differentiation into the oligodendrocyte lineage,promote outgrowth of the desired cells, inhibit growth of other celltypes, or perform any combination of these activities.

This section is a guide that illustrates to the reader some approachesthat can be taken to obtain oligodendrocytes of this invention. Exceptwhere otherwise required, explanations as to the underlying mechanism ofthe process are provided only as working hypotheses to assist in furtherelaboration. It is not necessary that the user understand thehypothesis, nor is it required that the invention conform to thehypothesis in order to be put into practice. Now the applicants havedemonstrated that oligodendrocytes can be made from pluripotent stemcells, further adjustments to the protocols can be made, and alternativemethods can be found to obtain the novel products described in thisdisclosure.

Steps to generate oligodendrocytes from pPS cells can include: a)obtaining a population of cells committed to make oligodendrocytes; b)expansion of oligodendrocyte lineage cells; and c) further maturation ofcells to late-stage oligodendrocytes.

Directing Stem Cells into the Oligodendrocyte Lineage

The process of generating oligodendrocytes typically involves twoaspects: causing the originating stem cell population to differentiate,and causing oligodendrocyte lineage cells to become the predominant celltype. These events can occur sequentially or concurrently.

The process of differentiation can be induced by causing the pPS cellsto form embryoid bodies or aggregates: for example, by overgrowth of adonor pPS cell culture, or by culturing pPS cells in suspension inculture vessels having a substrate with low adhesion properties thatallows embryoid bodies to form. In an exemplary method, confluentmonolayer cultures of hES cells are harvested and then plated innon-adherent cell culture plates, keeping the cells in suspension, andproviding regular feeding with nutrient medium.

Alternatively or in addition, the differentiation process can beinitiated by culturing with certain factors that prevent the cells frommaintaining the undifferentiated phenotype. The initial differentiationfactors need not limit differentiation into the oligodendrocyte lineage,but should be inclusive of oligodendrocytes or their precursors withinthe range of cell types in the differentiated population. Exemplarygrowth factors of this type are ligands that bind retinoid receptors, oractivate the extracellular-signal-regulated kinase (ERK) pathway. Amitogen such as a fibroblast growth factor (or those listed in the nextsection) is typically included in the culture medium to promoteproliferation while the differentiation process is under way.

At some stage, the culture can be directed more specifically into theoligodendrocyte lineage. This can be done by including in the culturemedium a factor that more specifically promotes the growth ofoligodendrocytes. Exemplary oligodendrocyte differentiation factors areligands and antibodies that bind thyroid hormone receptors on the cellsurface or in the nucleus, exemplified by T3(3,5,3′-triiodo-L-thyronine) and T4 (L-thyroxin) at about 40 ng/mL.Thyroid hormones are believed to increase expression of retinoic acidreceptors, and otherwise promote differentiation into cells of theoligodendrocyte lineage.

Another oligodendrocyte differentiation factor is selenium, anantioxidant that is believed to participate in the upregulation ofmyelin genes in differentiating oligodendrocytes. Other candidatedifferentiation factors are other antioxidants like Vitamin E, andfactors that increase activity of enzymes for which selenium is acofactor, such as thioredoxin reductase and the family of iodothyroninedeiodinases. Selenium is especially effective when included in theculture medium at relatively high concentrations of at least 20 ng/mL or100 ng/mL, in the form of selenite ion (SeO32-). Other candidatedifferentiation factors or cofactors include bone morphogenic protein(BMP), sonic hedge hog (SHH), and leukemia inhibiting factor (LIF). Alsocontemplated are combinations of oligodendrocyte differentiation factorsproduced by neural tissue, which can be generated by coculture withselected dissociated neural tissue or cell lines, exemplified byembryonic CD-1 mouse brain, and factor-containing extracts obtained fromthe tissue or cell line.

It has been discovered that oligodendrocytes can be generated in asurprisingly efficient manner by combining all of these techniquestogether: initiating differentiation by forming embryoid bodies in thepresence of a mitogen, general differentiation factors like retinoicacid, and specific differentiation factors like thyroid hormone andsodium selenite. Non-limiting illustrations provided in the Examplesection include these factors and other standard culture ingredients,such as nutrient supplements, growth factors such as insulin, andantibiotics. By including a suitable combination of differentiationfactors at the onset of differentiation, the culture will rapidlyproduce cells more committed to the oligodendrocyte lineage.

Expanding Oligodendrocyte Lineage Cells

As an optional step before using the cells for a desired purpose, theuser of this invention may want to increase the number ofoligodendrocyte lineage cells by causing them to proliferate further inculture.

This can be accomplished by culturing the cells in a nutrient medium inthe presence of one or more mitogens. Exemplary are members of thefamily of fibroblast growth factors, such as FGF-2 (basic FGF), andFGF-4. Also exemplary is epidermal growth factor (EGF), functionalhomologs, and other factors that bind the EGF receptor. Other candidategrowth factors are platelet-derived growth factor (PDGF), insulin-likegrowth factor (IGF), and factors that elevate cyclic AMP levels, such asforskolin.

In the presence of such mitogens, differentiation factor initiators suchas retinoic acid can be withdrawn. Since mitogens cause cells to grownon-specifically, it is often beneficial to continue includingoligodendrocyte-specific differentiation factors in the medium, tomaintain preferential growth of oligodendrocyte lineage cells.Preferential growth of oligodendrocytes can also be assisted byadjusting the balance of mitogens in the culture. For example, EGFmaintains ectoderm cells but not fibroblasts, which may die when FGF isno longer present. Once the cells that depend strictly on FGF have beeneliminated, then FGF can be added back into the culture to accelerategrowth of the desired cell type.

As another optional step in preparing the cells, the user of thisinvention may wish to separate oligodendrocyte lineage cells from othercell types that may be present in the culture. Various separationprocedures are contemplated, such as antibody or lectin mediatedadherence or sorting for cell surface markers. Suitable phenotypicmarkers for both positive and negative selection are listed below. Alsocontemplated are sorting of oligodendrocyte lineage cells usingpromoter-reporter plasmids, constructed with tissue-specific promoterssuch as those for UDP-galactose ceramide galactosyltransferase (CGT),2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP), NG2 proteoglycan,NCAM, myelin basic protein (MBP), or various myelin-associated proteins;linked to a reporter gene such as alkaline phosphatase, greenfluorescent protein, or luciferase.

It has been discovered that oligodendrocyte lineage cells can beseparated from other cells that propagate in the presence ofoligodendrocyte differentiation factors in a more simple fashion byadhering the cells to a suitable substrate. Oligodendrocytes bearcell-specific carbohydrates and cell-surface receptors, and willpreferentially adhere to the conjugate ligand. In particular,oligodendrocytes can be separated by adherence to certain basementmembrane components, such as laminin, gelatin, or Matrigel®, acommercially available extracellular matrix preparation fromEngelbreth-Holm-Swarm tumor cells that gels at room temperature to forma reconstituted basement membrane (Example 1). Once oligodendrocyteshave adhered to the matrix (a few hours to a few days), other cell typescan be washed away, and the adherent cells recovered, for example, bybrief trypsin digestion. Besides enriching for the desired cell type,this procedure has the advantage of breaking up larger agglomerates ofcells for ease of further manipulation.

Effectiveness of separation procedures can be determined by measuringenrichment for the markers listed below. Once the oligodendrocytes havebeen purified in this fashion, they can be expanded further in thepresence of mitogens as already described, matured as described in thefollowing section, or formulated in a manner suitable for ultimate use.

Further Maturation

When desired, the cells of this invention can be further matured beyondthe replicative phase into a functional phenotype. This is may be doneto characterize the potential of the progenitors, or obtain end-stagecells for therapeutic or drug screening purposes.

Maturation is effected by changing the growth conditions in a mannerthat inhibits further proliferation of the precursor phenotype. Forexample, the cells can be plated onto a substrate such as poly-L-lysinethat promotes emergence of the mature phenotype. Alternatively or inaddition, one or more of the growth factors used to expand the cells iswithdrawn. Factors that enhance maturation can be included in thematuration medium, such as ciliary neurotrophic factor (CNTF), and otheragonists of the CNTF receptor. Once the cells have reached the desiredmaturity, they can be harvested from culture (for example, using trypsinor collagen) and formulated for analysis or ultimate use.

Characteristics of Differentiated Cells

Cells can be characterized according to a number of phenotypic criteria.The criteria include but are not limited to microscopic observation ofmorphological features, detection or quantitation of expressed cellmarkers, functional criteria measurable in vitro, and behavior uponinfusion into a host animal.

Phenotypic Markers

Differentiated cells of this invention can be characterized according towhether they express phenotypic markers characteristic ofoligodendrocytes. Classic immunocytochemical markers for oligodendrocytecells that may be present depending on the maturity of the cellpopulation are the following:

-   -   NG2, a chondroitin sulfate proteoglycan expressed by macrophages        and oligodendrocyte progenitors    -   galactocerebroside (GalC), a marker for committed        oligodendrocytes    -   myelin basic protein (MBP), a marker of mature myelin and        myelin-producing cells

Other useful markers expressed by oligodendrocyte lineage cells includethe following:

-   -   PDGFRα, a membrane receptor for PDGF, expressed by        oligoprogenitors, oligodendrocytes, and other cell types    -   TRα1, an nuclear receptor for thyroid hormone, expressed by        oligoprogenitors, oligodendrocytes, neurons, and other cell        types    -   myelin proteolipid protein, a component of myelin that is        expressed on oligodendrocytes and glial precursors    -   the epitope defined by O4 antibody, a marker for        oligodendrocytes, astrocytes, and their precursors    -   vimentin, a fibroblast-type filament protein that marks        astrocyte precursors (often negative on oligodendrocytes)    -   glial fibrillary acidic protein (GFAP), a marker for astrocytes        (negative on oligodendrocytes)    -   A2B5, an epitope expressed on type II astrocytes, glial        progenitors, oligodendrocyte progenitors, and pancreatic β cells    -   the epitope recognized by RIP antibody, which stains        oligodendrocytes and their processes, and coincides with        myelinated axons in both the spinal cord and the cerebellum

Transcription factors expressed at various times during the pathway ofoligodendrocyte differentiation include the following:

-   -   Olig1, a helix-loop-helix (HLH) family transcription factor,        expressed by oligoprogenitors, motor neuron progenitors, and        kidney cells    -   Olig2, another HLH family transcription factor, expressed by        oligoprogenitors, motor neuron progenitors, and pineal gland    -   Sox10, a Sox family transcription factor, expressed by        oligoprogenitors, oligodendrocytes, Schwann cells, neural rest,        cochlea, prostate, and melanocytes    -   Nkx2.2, a Hox family transcription factor, expressed by        oligoprogenitors, oligodendrocyt s, neuronal progenitors,        pancreatic α and β cells    -   Pax6, an HLH family transcription factor, expressed by        oligoprogenitors, neuronal progenitors, pancreatic α and β        cells, lens retina, pituitary, liver, and spleen

Useful markers for other cell types include the following:

-   -   neuronal nuclear antigen (NeuN), a marker of neuronal maturation        (normally negative in oligodendrocyte lineage cells)    -   class III beta-tubulin (TuJ1), another marker of neuronal cells    -   microtubule associated protein 2 (MAP-2), a marker for CNS cells        (may be positive)    -   SSEA-4, Oct-4, and telomerase reverse transcriptase (TERT),        markers for undifferentiated pPS cells (negative on        oligodendrocytes and their precursors)

Tissue-specific markers can be detected using any suitable immunologicaltechnique—such as flow immunocytochemistry for cell-surface markers, orimmunohistochemistry (for example, of fixed cells or tissue sections)for intracellular or cell-surface markers. A detailed method for flowcytometry analysis is provided in Gallacher et al., Blood 96:1740, 2000.Expression of a cell-surface antigen is defined as positive if asignificantly detectable amount of antibody will bind to the antigen ina standard immunocytochemistry or flow cytometry assay, optionally afterfixation of the cells, and optionally using a labeled secondary antibodyor other conjugate to amplify labeling.

The expression of tissue-specific gene products can also be detected atthe mRNA level by Northern blot analysis, dot-blot hybridizationanalysis, or by reverse transcriptase initiated polymerase chainreaction (RT-PCR) using sequence-specific primers in standardamplification methods. See U.S. Pat. No. 5,843,780 for further details.Sequence data for particular markers can be obtained from publicdatabases such as GenBank. For example, myelin galactolipid biosyntheticenzyme UDP-galactose ceramide galactosyltransferase (CGT, GenBankAccession No. AH006651) is the enzyme that catalyzes the final step inthe synthesis of GalC.

To facilitate use in research or therapy, it is often beneficial tomaximize the proportion of cells in the population that have thecharacteristics of oligodendrocytes or their precursors. As illustratedin the Example section below, it is possible to obtain populations ofcells that are anywhere from at least about 20%, to over about 60%, 80%,90%, 95%, or even 98% oligodendrocyte precursors or mature cells (or amixture of both), identified as being positive for one, two, three, ormore of any of the phenotypic markers characteristic of such cells, asillustrated above.

For therapeutic applications relating to reconstitution of neuralfunction, it is often desirable to minimize the ability of the cellpopulation to form other cell types—particularly undifferentiated pPScells, and cells of non-ectodermal lineage. Depending on theapplication, it may also be advantageous to minimize the proportioncells of the neuronal lineage and their committed precursors, cells ofthe astrocyte lineage and their committed precursors, and commonprecursors for glial cells or all types of neural cells. In certainembodiments, oligodendrocyte populations according to this inventionhave less than ˜1%, 0.2%, or 0.05% contamination with these other typesof cells.

The skilled reader will already appreciate morphological features thatare characteristic of oligodendrocyte lineage cells. Oligodendrocyteprecursors sometimes take a bipolar shape, having two processesextending off opposite poles from the central body. They may also takethe form of relatively flat cells, bearing many of the same markers andother characteristics of oligodendrocytes. They are distinguishable fromother cells (like fibroblasts) by relatively slow growth rates anddependence on extracellular matrix and soluble factors. Bipolar and flatcells may be interconvertable, depending on the presence or absence ofgrowth factors (such as EGF, bFGF) that promote predominance of flatcells. Precursor populations of this invention may comprise at least˜20%, 40%, 60%, 80% or more of the bipolar or the flat cell phenotype.

Upon differentiation to more mature oligodendrocytes, the number andcomplexity of the processes will typically increase. The processes mayappear to have myelin webbing in between, which wrap around individualaxons to form myelin sheath that promotes neural transmission along theaxon.

Genotypic Features

When derived from an isolated pPS cell, or an established line of pPScells, the oligodendrocytes of this invention can be characterized asbeing the progeny of the originating cell or cell line. Accordingly, theoligodendrocytes will have the same genome as the cells from which theyare derived. This means that over and above any karyotype changes, thechromosomal DNA will be over 90% identical between the pPS cells and theoligodendrocytes. Oligodendrocytes that have been treated by recombinantmethods to introduce a transgene or knock out an endogenous gene arestill considered to have the same genome as the line from which they arederived, since all non-manipulated genetic elements are preserved.

Oligodendrocytes and pPS cells can be identified as having the samegenome by standard genetic fingerprinting techniques. Possession of thesame genome can also be inferred if the oligodendrocytes are obtainedfrom the undifferentiated line through the course of normal mitoticdivision.

In certain industrial applications, this characteristic is a valuablefeature of the oligodendrocytes of this invention. In particular, theavailability of the originating pPS cells provides a further supply ofgenetically matched oligodendrocytes, since the pPS cells can be causedto proliferate, subdivided, and differentiated into moreoligodendrocytes as required. Furthermore, the pPS cells can bedifferentiated into other therapeutically important lineages. Forexample, they can be differentiated into immunotolerizing cellpopulations that can help render the intended recipient tolerant toengraftment with matched oligodendrocytes (International applicationPCT/US01/43434, Geron Corp.).

The techniques described in this application allow large populations ofoligodendrocyte lineage cells to be produced that share the same genome,by expanding the cells before or after differentiation. Populations of108, 1010, or 1012 cells are theoretically possible. Such largepopulations are usually divided into separate containers suitable forfurther culture, drug screening, or therapeutic administration.

Certain embodiments of the invention include originating cells (such asa undifferentiated pPS cell line, or an intermediate population) incombination with differentiated cells bearing characteristics of glialcells or oligodendrocytes. The two populations may either be in the samecontainer, in separate containers in the same facility, or in twodifferent locations. The undifferentiated and differentiated cells maybe present simultaneously or at a different time, such as when a cultureof undifferentiated cells is caused to differentiate it its entiretyinto oligodendrocyte lineage cells, as already described.

Functional Features

For purposes of quality control, it is often desirable to characterizethe features of the differentiated cells of this invention by functionalcriteria. Different functions may be of different relative interestdepending on the intended ultimate use. For example, the cells can beassessed for their ability to remyelinate neuronal tissue in tissueculture, to repair sites of induced demyelination in vivo, or theirability to restore neurological function in an injured subject. A numberof experimental models exist for determining these features, includingthe following non-limiting illustrations.

1. In Vitro Myelination In Coculture: Adult dorsal root ganglion (DRG)cultures are prepared by harvesting lumbar and cervical DRG neurons from2-3 month old female rats. The cells are triturated, centrifuged, andresuspended in DMEM F12 medium at ˜100,000 viable neurons per mL, andthen plated onto laminin coated dishes for 4 weeks. In vitro myelinationis conducted by adding ˜2.5×10⁴ oligodendrocyte lineage cells, andculturing an additional 4 weeks with daily feeding. Myelination ofrodent axons by human oligodendrocytes is illustrated in Target &Blakemore, Eye 8(part 2):238, 1994.

To determine the ability of oligodendrocyte lineage cells to formmyelin, the cocultures are fixed with 4% paraformaldehyde overnight andstained for GalC. Visual fields adjacent to the ganglion are selected,and scored for the number of myelin segments per field. Differentiationpotential is determined by staining low-density cultures for markerssuch as nestin, β-tubulin III, GFAP, CNP, GalC, Ki-67 antigen, NeuN, orneurofilament 70.

Evidence for myelination of the DRG in culture generally correlates withbiological efficacy of the oligodendrocytes. Ability of the cells tosurvive and enhance myelination or axonal regrowth in vivo can befurther obtained using a suitable animal model. On the other hand,myelination in coculture will depend on the conditions of the culture,and a negative result does not exclude efficacy in vivo. The predictivevalue of the in vitro test can be improved by adjusting the cultureconditions: for example by adding factors believed to enhancemyelination, such as IGF, or a neurotrophin like NT-3.

2. Model for Chronic Demyelination: Regions of chronic demyelination canbe induced in the adult rat dorsal column (Keirstead et al., J.Neurosci. 19:7529, 1999). The spinal cord is exposed to 40 G ofX-irradiation over a distance of 2 cm centered on T9 using leadshielding, which introduces nicks into the DNA of exposed cells, whichcauses death of the cells that are dividing. This is followed by directintraspinal injection of ethidium bromide 2 days later at T9. Ethidiumbromide is a DNA interchelating agent that kills cells exposed to it,rendering an acellular region of chronic demyelination and free ofviable oligodendrocytes and astrocytes through ˜60% of the area of thedorsal column.

Three days later, the animals receive transplants of oligodendrocytelineage cells into the site of demyelination. Optionally, the cells canbe prelabeled with bromodeoxyuridine (BrdU) added to the culture medium48 h in advance. In the first instance, cells can be prepared asclusters of ˜30 precursors, concentrated to a density of ˜60,000 cellsper μL. One μL of cells is administered into the injury site using apulled glass micropipette of ˜80 um outside diameter over ˜10 min. Afterabout 2-4 weeks, tissue samples are prepared for resin or cryostatsectioning into 1 mm transverse blocks.

Sections from the coronal face are stained with toluidine blue andanalyzed for general pathology, evidence of remyelination, and cellmorphology. Since the induced regions are acellular, cells present aftertransplantation are derived from the administered cells. Cryostatsections can be stained for markers of relevant cell types, such as GFAP(astrocytes), CNP (oligodendrocytes), RIP (oligodendrocytes), or NeuN(neurons). Ultrathin sections can also be analyzed by electronmicroscopy for number of myelin lamellae and cell ultrastructure.Redistribution of transplanted cells throughout demyelination, anddifferentiation into mature myelinating cells can be determined.Remyelination as a percentage of demyelinated axons at a level of about25%, 50%, or 75% is evidence of increased biological efficacy.

Therapeutic capability of oligodendrocyte lineage cells can also betested in congenital models of dysmyelination. Established modelsinvolving a mutation or defect in myelin basic protein include theshiverer mutant mouse (Roach et al., Cell 42:149, 1985), and the LongEvans shaker rat (Kwiecien et al., J. Neurocytol. 27:581, 1989; Delaneyet al., Lab. Anim. Sci. 45:547, 1995). Reconstruction can be tested byintracerebroventricular or cisternal transplantation (Mitome et al.,Brain 134:2147, 2001), or by administration directly into the spinalcord (Liu, McDonald et al., Proc. Natl. Acad. Sci. USA 97:6126, 2000).Formation of compact myelin in models involving a mutation or defect inMBP is directly attributable to the administered cells, since theseanimals are normally not capable of proper myelination, and sufferneurological deficits as a result.

3. Model for Spinal cord Injury: Models for SCI include contusioninjuries and dorsal hemisection. For contusion injuries, the spinalcourse is displaced for ˜0.9 mm (moderate injury) over 23 msec using asuitable spinal contusion injury device. For hemisection injuries, thedorsal half of the spinal cord is cut with a pointed scalpel blade usinga stereotactic manipulator. Both procedures are followed by suitablepostoperative care.

To promote migration of implanted cells and remove myelin-associatedgrowth inhibitors, the spinal cord is optionally also demyelinated(Keirstead et al., Exp Neurol. 151:303, 1998). A 2 mm hole is producedin the center of the vertebra canal and caudal to the site of axonalinjury. The exposed spinal cord is the injected with ˜4 μL polyclonalanti-GalC antibody (Chemicon) at a dilution of 1:2 with 33% guinea pigcomplement (Harlan SeraLab) in phosphate buffered saline.

The animals are transplanted with oligodendrocyte lineage cells ˜24 hafter injury through a pulled glass micropipette. Alternatively, achronic injury model can be created by withholding treatment for 1-3months. Following treatment with the cells, functional response can berecorded by video tape, and monitored on a regular basis for evidence ofclinical improvement. For example, overground locomotion can bequantitated using the BBB scale, a 21-point scale based on jointmovements, weight support, limb coordination, and other features(Example 2).

For histological workup, the animals can be preinjected with tracerssuch as biotin-labeled cholera toxin β-subunit (CTB, 4 μL of 0.1%bilaterally into sciatic nerves), or biotinylated dextran amine (BDA, 10μL of 10% into the sensorimotor cortex). About 2 weeks later, tissuesections are prepared as in the demyelination models for location oftransplanted cells and evidence of remyelination, which isdistinguishable from normal myelination or partial demyelination bymorphological criteria. Sections are also analyzed for axons labeledwith the injected tracers, for evidence of characteristic neurite growthcones. Immunocytochemical evidence of axonal regeneration can beassessed by staining for neuronal markers such as RT97 antigen (a markerof neurofilament), serotonin (5HT), norepinephrine (NE), and calcitoningene-related peptide (CGRP).

Genetic Modification of Oligodendrocytes

Certain oligodendrocyte precursor populations of this invention havesubstantial proliferation capacity. If desired, the replication capacitycan be further enhanced by increasing the level of telomerase reversetranscriptase (TERT) in the cell, by either increasing transcriptionfrom the endogenous gene, or introducing a transgene. Particularlysuitable is the catalytic component of human telomerase (hTERT),provided in International Patent Application WO 98/14592. Transfectionand expression of telomerase in human cells is described in Bodnar etal., Science 279:349, 1998 and Jiang et al., Nat. Genet. 21:111, 1999.

Telomerase expression can be increased in undifferentiated pPS cells,which can then be differentiated into oligodendrocytes according to thisinvention. Alternatively, the pPS cells can be differentiated intooligodendrocyte precursors, and then transfected to increase TERTexpression. Genetically altered cells can be assessed for TERTexpression by RT-PCR, telomerase activity (TRAP assay),immunocytochemical staining for TERT, or replicative capacity, accordingto standard methods. Other methods of immortalizing cells are alsocontemplated for certain applications, such as transforming the cellswith DNA encoding myc, the SV40 large T antigen, or MOT-2 (U.S. Pat. No.5,869,243, International Patent Applications WO 97/32972 and WO01/23555).

If desired, the cells of this invention can be prepared or furthertreated to remove undifferentiated cells in vitro, or to safeguardagainst revertants in vivo. One way of depleting undifferentiated stemcells from the population is to transfect the population with a vectorin which an effector gene under control of a promoter that causespreferential expression in undifferentiated cells—such as the TERTpromoter or the OCT-4 promoter. The effector gene may be a reporter toguide cell sorting, such as green fluorescent protein. The effector maybe directly lytic to the cell, encoding, for example, a toxin, or amediator of apoptosis, such as caspase (Shinoura et al., Cancer GeneTher. 7:739, 2000). The effector gene may have the effect of renderingthe cell susceptible to toxic effects of an external agent, such as anantibody or a prodrug. Exemplary is a herpes simplex thymidine kinase(tk) gene, which causes cells in which it is expressed to be susceptibleto ganciclovir (U.S. Pat. No. 6,576,464 B1). Alternatively, the effectorcan cause cell surface expression of a foreign determinant that makesany cells that revert to an undifferentiated phenotype susceptible tonaturally occurring antibody in vivo (U.S. 2003-0032187 A1).

The cells of this invention can also be genetically altered in order toenhance their ability to be involved in tissue regeneration, or todeliver a therapeutic gene to the subject being treated. A vector isdesigned using the known encoding sequence for the desired gene,operatively linked to a promoter that is either constitutive (such asthe CMV promoter) or specifically active in cells of the oligodendrocytelineage cells (such as the promoter for myelin basic protein). Varioustransgenes may be expressed according to this strategy, such as thosethat enhance oligodendrocyte growth, activate remyelination, or promoteaxon regeneration. Exemplary are genes encoding nerve growth factors(U.S. Pat. Nos. 5,885,584 and 6,268,340).

Use of Oligodendrocytes in Research and Clinical Therapy

This invention provides a method to produce large numbers ofoligodendrocytes for a variety of important research, development, andcommercial purposes.

The cells of this invention can be used to prepare a cDNA libraryrelatively uncontaminated with cDNA preferentially expressed in cellsfrom other lineages. The differentiated cells of this invention can alsobe used to prepare monoclonal or polyclonal antibodies that are specificfor markers of oligodendrocytes and their derivatives, according tostandard methods.

Of particular interest are use of the compositions of this invention fordrug development and clinical therapy.

Drug Screening

The cells of this invention can be used to screen for factors (such assolvents, small molecule drugs, peptides, polynucleotides) orenvironmental conditions (such as culture conditions or manipulation)that affect the characteristics of both oligodendrocyte precursors andmature oligodendrocytes.

In one example, pPS cells (undifferentiated or initiated into thedifferentiation paradigm) are used to screen factors that promotematuration into oligodendrocytes, or promote proliferation andmaintenance of oligodendrocytes in long-term culture. For example,candidate maturation factors or growth factors are tested by adding themto cells in different wells, and then determining any phenotypic changethat results, according to desirable criteria for further culture anduse of the cells. This can lead to improved derivation and culturemethods not only for pPS derived oligodendrocytes, but also foroligodendrocytes and their progenitors isolated from primary neuraltissue.

Other screening methods of this invention relate to the testing ofpharmaceutical compounds for a potential adverse effect onoligodendrocyte growth, development, or toxicity. This type of screeningis appropriate not only when the compound is designed to have apharmacological effect on oligodendrocytes themselves, but also to testfor oligodendrocyte-related side-effects of compounds designed for aprimary pharmacological effect elsewhere.

Other screening methods relate to the use of oligodendrocytes to measurethe effect of small molecule drugs that have the potential to affectoligodendrocyte activity in their role of myelinating axons. To thisend, the cells can be combined with test compounds in vitro, and theeffect of the compound on myelin related activities is determined—forexample, expression of myelin-associated components, such as myelinbasic protein; formation of myelin sheaths detectable histologically,and the ability to myelinate neighboring axons in cocultures withneuronal cells.

For general principals of drug screening, the reader is referred to U.S.Pat. No. 5,030,015, and to the textbook In vitro Methods inPharmaceutical Research, Academic Press 1997. Assessment of the activityof candidate pharmaceutical compounds generally involves combining thedifferentiated cells of this invention with the candidate compound,either alone or in combination with other drugs. The investigatordetermines any change in the morphology, marker phenotype, or functionalactivity of the cells that is attributable to the compound (comparedwith untreated cells or cells treated with an inert compound), and thencorrelates the effect of the compound with the observed change.

Oligodendrocytes in Clinical Therapy

This invention provides for the use of oligodendrocyte precursor cellsand their derivatives to retain or restore neural function in a patientin need of such therapy. In particular, the cells of this invention maybe administered in order to remyelinate neuronal tissue, or otherwiseprovide support for the maintenance or regeneration of neural networks.Without implying any limitation, the administered cells may have theeffect of stabilizing or improving the function of neurons already inplace, or assist neurons in forming new connections with each other orthe tissues they control.

The nature of the pharmaceutical composition will depend in part on thecondition to be treated. In some instances, it may be appropriate toformulate the composition with precursor cells (positive for NP2 orGalC) having good replicative capacity and resilience to manipulation.In other instances, it may be appropriate to formulate the compositionwith more mature cells (positive for GalC or MBP) to provide moreimmediate myelination capacity.

It has been discovered that relatively disperse populations ofoligodendrocyte lineage cells can be obtained easily from pPS derivedcells by adhering large aggregates that form in suspension culture to asuitable matrix, such as Matrigel®. The cultured cells are plated on thesubstrate for a few hours to a few days, and non-adherent cells areremoved. Oligodendrocyte lineage cells are then recovered by a suitablechemical or mechanical means, such as brief enzymatic digestion followedby trituration. Monodisperse populations or clusters no more than ˜30cells are believed to have advantages in terms of cell manipulation,storage characteristics, and ability to provide a beneficial effect uponadministration. The cells are typically washed, and then suspended in apharmaceutically compatible medium at a concentration suitable foradministration of an effective dose so as to retain the cells at thedisease site (say, 20,000 to 100,000 cells per μL, scaled by the volumeof the treatment area).

Patients are prepared according to the usual procedures for neurologicaladaptive therapy. The patients may be treated to prevent rejection usingstandard immunosuppressive therapy, such as Cyclosporin A. Alternativelyor in addition, they may be specifically tolerized to the allotype ofthe oligodendrocytes using hematopoietic cells or undifferentiated cellsprepared from the same pPS cell line (WO 02/44343; WO 03/050251). Insome instances, it may be beneficial to transiently demyelinate neuronsat the injury site, to improve access of the administered cells orremove factors that could inhibit remyelination. One means to accomplishthis is to administer to the local environment complement-fixingantibodies to one or more epitopes on the myelin sheath, such as GalC,04, or myelin associated glycoprotein. See Keirstead et al., Brain Res.Bul. 44:727, 1997; and Canadian Patent 2,253,078. The differentiatedcells of this invention are then administered in one or more doses at oraround the site where myelination or regeneration of neurologicalfunction is desired.

The subject then receives supportive post-operative care, and ismonitored for graft acceptance or regeneration of neurological function.Where appropriate, patients may be concurrently treated with other meansbelieved to restore oligodendrocyte function, such as Fampridine-SR(4-aminopyridine). Histological samples can be analyzed byimmunocytochemistry for relevant markers as already described, andassessed for functional events, such as axon remyelination, and neuritesprouting caused by the presence of the administered cells. The patientscan be monitored for maintenance or improvement in their clinicalcondition, according to the typical pathological features of theirdisease, and assessment of function, for example, on the expandeddisability status scale (EDSS).

Conditions suitable for treatment with the compositions of thisinvention include but are not limited to conditions involvingprogressive demyelination, and trauma of the central nervous systemwhere the ability to maintain or produce myelin may either contribute tohealing, or help prevent further deterioration.

Multiple Sclerosis is a slowly progressive disease characterized bydisseminated patches of demyelination in the brain and spinal cord.Plaques of demyelination, with destruction of oligodendroglia andperivascular inflammation, occur throughout the CNS, primarily in thewhite matter (especially in the cervical and dorsal regions), the opticnerves, and periventricular areas. Tracts in the midbrain, pons, andcerebellum, and gray matter in the cerebrum and spinal cord are alsoaffected.

Acute disseminated encephalomyelitis (postinfectious encephalomyelitis)is characterized by perivascular CNS demyelination, which can occurspontaneously but usually follows a viral infection or viralvaccination. Chronic inflammatory demyelinating polyradiculoneuropathy(CIDP) is characterized by interstitial and perivascular infiltration ofthe endoneurium with inflammatory T cells and macrophages, leading tosegmental demyelination of peripheral nerves. HTLV-associated myelopathyis a slowly progressive spinal cord disease characterized by spasticweakness of both legs. Some peripheral neuropathies, such asGuillain-Barre syndrome, are also characterized by demyelination.

Congenital metabolic disorders (such as phenylketonuria and otheraminoacidurias; Tay-Sachs, Niemann-Pick, and Gaucher's diseases;Hurler's syndrome; Krabbe's disease and other leukodystrophies) affectthe developing myelin sheath in the CNS, and may cause permanentwidespread neurologic deficits. Adrenoleukodystrophy andadrenomyeloneuropathy are X-linked recessive metabolic disorderscharacterized by adrenal gland dysfunction and widespread demyelinationof the nervous system. Pelizaeus-Merzbacher disease is a condition wherepatents do not form myelin because of a point mutation in theproteolipid protein gene. Leber's hereditary optic atrophy and relatedmitochondrial disorders are characterized primarily by bilateral loss ofcentral vision.

Any acute or long-term abnormalities caused by trauma to the CNS may beconsidered for treatment, as well as conditions related to loss ofmyelin through anoxia and ischemia. Included in this category areconditions related from stroke or traumatic brain injury.

Spinal cord injuries of various kinds, causing paraplegia or incompletemotor function at any level, are prime candidates for treatment usingthe cells of this invention. Depending on the nature and accessibility,injuries to the cervical, lumbar, thoracic, and sacral spine may allderive an improvement or stabilization of their condition. Acuteinjuries causing complete or incomplete loss of SC function can betreated simultaneously or soon after decompression surgery. Chronicconditions can be treated or retreated whenever desirable. The cells canbe administered at or near the site of the injury by surgery, endoscopy,or percutaneous injection, using a needle, cannula, or other suitabledevice. If warranted, the electrophysiological effect can be determinedusing an evoked potentials test. The patients can also be assessed forclinical improvement in the motor and sensory pathway by criteria suchas the American Spinal Injury Association (ASIA) motor score, and theFerrans and Powers Quality of Life Index (QLI).

As always, the ultimate responsibility for patient selection, mode ofadministration, and monitoring of rehabilitation is the responsibilityof the managing clinician.

For purposes of commercial distribution, oligodendrocytes of thisinvention are typically supplied in the form of a pharmaceuticalcomposition comprising an isotonic excipient, and prepared underconditions that are sufficiently sterile for human administration. Thisinvention also includes a reagent system, comprising a set orcombination of cells that exist at any time during manufacture,distribution, or use. The cell sets comprise any combination of two ormore cell populations described in this disclosure, exemplified but notlimited to a type of differentiated pPS-derived cell (glial cells,oligodendrocytes, their precursors and subtypes, and so on), incombination with undifferentiated pPS cells or other differentiated celltypes, sometimes sharing the same genome. Each cell type in the set maybe packaged together, or in separate containers in the same facility, orat different locations, at the same or different times, under control ofthe same entity or different entities sharing a business relationship.

For general principles in medicinal formulation of cell compositions,the reader is referred to Cell Therapy: Stem Cell Transplantation, GeneTherapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds,Cambridge University Press, 1996; and Cell Transplantation forNeurological Disorders, T. B. Freeman et al. eds., Humana Press 1998.The cells may be packaged in a device or container suitable fordistribution or clinical use, optionally accompanied by informationrelating to the storage of the cells or their use as a medicament totreat the clinical conditions referred to above, or for any otherworthwhile purpose.

The following examples are provided as further non-limitingillustrations of particular embodiments of the invention.

EXAMPLES Example 1 Differentiation of hES Cells into Oligodendrocytes

Human embryonic stem (hES) cells of the H7 and H1 lines were propagatedin the undifferentiated state in feeder-free conditions on a Matrigel®substrate in knockout (serum-free) medium conditioned by primary mousefeeder cells and containing added bFGF (WO 99/20741; WO 01/51616, GeronCorp).

The hES cells were differentiated into cells of the oligodendrocytelineage according to the following scheme: TABLE 1 OligodendroprogenitorProduction from hES cells Day: 1 2 3-10 11-15 15-28 28 29-35 35-36 37-4142 Culture Suspension Add Full Remove Remove Plate on Expand Plate onRemove Fix conditions: culture retinoic strength RA bFGF Matrigel ®cells PLL- growth cultures acid medium Laminin factors (RA) Medium: TRTR GRM GRM GRM GRM GRM GRM GRM GRM Additives: bFGF bFGF RA bFGF EGF EGFEGF bFGF — — 4 ng/mL 4 ng/mL; 10 μm 2 ng/mL; 20 ng/mL 20 ng/mL 20 ng/mL2 ng/mL; RA EGF EGF 10 μM 20 ng/mL 20 ng/mLGRM is Glial Restriction Medium (see Table 2).

Transition medium (TR) is made as a 1:1 mixture of GRM and conditionedmedium for making hES cells. TABLE 2 Ingredients of Glial RestrictionMedium (GRM) Manufacturer Stock Final Component (catalog) concentrationconcentration Quantity DMEM:F12 Gibco (10565-018) — — 500 mL (basemedium) B27 supplement Gibco (17504-044) 50× 1:50 10 mL Insulin Sigma(I-1882) 25 mg/mL 25 μg/mL 1 μL/mL Progesterone Sigma (P-6149) 65 μg/mL65 ng/mL 1 μL/mL Putrescine Sigma (P-6024) 10 mg/mL 10 μg/mL 1 μL/mLSodium selenite Sigma (S-9133) 0.1 mg/mL 100 ng/mL 1 μL/mL TransferrinSigma (T-8158) 50 mg/mL 50 μg/mL 1 μL/mL (Holotransferrin) T3(triiodothyronine) Sigma (T-67407) 40 μg/mL 40 ng/mL 1 μL/mL

The differentiation protocol was conducted as follows:

-   -   Day 1: Collagenase IV (Gibco 17101-015) was used to dissociate        the ES colonies from the adherent substrate and colonies were        placed in low adherent 6 well plates (Corning 3471) in TR with 4        ng/mL bFGF (Gibco 13256-029). Penicillin-Streptomycin (Gibco        10378-016) was used for 3 days only at the beginning of        differentiation protocol.    -   Day 2-10: Cells were cultured with retinoic acid (RA;        all-Trans-retinoic acid, Sigma 223018), 10 μM, feeding daily for        a total of 8 days. The RA stock solution was prepared in DMSO at        a concentration of 6 mg/mL (approx. 0.02 M).    -   From day 3, the medium was replaced with GRM, and no more bFGF        was added to the culture. Medium was replaced every day. During        feeding, light was dimmed to minimum because RA is light        sensitive. Medium from the wells containing the ES cell        aggregates was collected in 15 mL tubes. After a short        centrifugation at low speed (800 rpm, 1 minute), the supernatant        was aspirated and fresh medium was added. Gentle pipetting up        and down (2-3 times) assured a homogenous distribution of 4 mL        in each well of the 6 well plates.    -   Day 10 to 15: After day 10, EGF (Sigma E9644) at a concentration        of 20 ng/mL and bFGF 2 ng/mL was added during the daily feeding.    -   Day 15 to 21: bFGF was suppressed from culture medium, while EGF        was continued at a concentration of 20 ng/mL.    -   After day 21: New aggregates/clusters with neural        characteristics could be generated even after day 42 by        maintaining the GRM medium with 20 ng/mL EGF.    -   To select for these neurospheres, the entire culture was        transferred on the 28^(th) day of the differentiation procedure,        without using any dissociation protocol, on Matrigel® coated        6-wells plates (Matrigel® 1:30, BD Bioscience 356231) for 12-20        hours (overnight). The next day, after a gentle shake of the        culture, only the neurospheres remained adherent, and the rest        of culture was replaced with fresh GRM medium.    -   On day 35, the neural aggregates were plated on poly-L-lysine        (Sigma P2636) and laminin coated four chamber imaging slides        (Nalgene-Nunc International 154917). A short treatment (5 min)        with Trypsin-EDTA was used to dissociate the cell        agglomerations. The cells were grown for 7 days on the        PLL-laminin, during which cultures were fed every other day.    -   On day 42, the cultures were fixed using paraformaldehyde, and        the cells were characterized by immunohistochemistry. Markers        used: NeuN, Galactocerebroside (GalC), Map2, O4, Vimentin, GFAP.

Results are shown in the accompanying phase inverted photomicrographs.

FIG. 1 shows embryoid bodies cultured for two days in suspension intransition medium (50% glial precursor medium).

FIG. 2 shows spheres on day 7 of differentiation, at the end ofculturing in retinoic cid. From day 3 onwards, clear spheres beganappearing in the suspension culture. By day 7, these spheres constituted80-90% of the cells in the culture.

FIG. 3 shows collapse of spheres after suppression of RA treatment. Someof the vesicles continued to grow by addition of cells to the inside andoutside of the sphere; the cell conglomerations took a dark shadedspherical shape. The spheres collapsed in presence of 20 ng EGF alone.However, the spheres could be maintained by adding a low concentrationof bFGF. Some of the spheres continued to grow by addition of new cells,resulting a spherical shaped, dark shaded cell conglomeration. Somesmall spheres with thick walls continued to grow, but completelydisappeared after few days. Accordingly, the bFGF was retained in theculture at low concentration (2 ng/mL) for 5 days, and the spherescontinued to grow by adding layers.

FIG. 4 shows the changes appearing after bFGF was removed. Most of thecell conglomerates started to dissociate and the entire culture mediumwas flooded with single cells and small clusters. At the same time, newbright yellow spheres were observed (arrows).

FIG. 5 shows that the bright-yellow spheres (arrows) were selectivelymaintained as the culture continued. New spheres appeared every day,some budding from other spheres, some from the isolated cell mass andsome from the dark cell agglomerations.

On day 28, the entire culture was adhered to a Matrigel® substrateovernight. Only the neural committed spheres adhered to substrate, whilethe rest of the culture continued to float.

FIG. 6 shows migration and branching of the glial committed neuralprecursors from the spheres 2-3 days after plating on Matrigel®. Thesmaller colonies formed a circle by longitudinal division of individualcells, while the large ones showed a star shape migration with longprocesses. More migrating cells appeared after a few days of growth onMatrigel®).

After a week, the culture was dissociated from Matrigel® using Trypsin,and then plated on poly-L-lysine and Laminin (Gibco 12163-010) coatedNunc imaging chambers at low density for further characterization. Theplated cells adopted characteristically oligodendrocyte shapes after 3-4days of growth on Laminin substrate.

It was subsequently discovered that the Matrigel® selection step can bereduced to 10-20 hours. The non-adherent cells are discarded, and theadherent cells are then resuspended and expanded in the presence of FGF,EGF, and glial precursor medium. This has the advantage of producing amore disperse cell population more suited for therapeutic administrationand other purposes.

FIG. 7 shows the results of this technique. After adhering to Matrigel®,the cells were expanded for 7 days, plated onto poly-L-lysine laminin,and cultured in the absence of mitogens. The cells were fixed with 4%paraformaldehyde, and blocked in 3% goat serum and 0.3% Triton-X 100®detergent. Immunocytochemistry was performed using antibody togalactocerebroside (GalC, Chemicon), followed by peroxidase-labeledanti-immunoglobulin. Nuclei were counterstained using hematoxin. (Firstpanel 20× magnification; second panel 40× magnification).

At least about 95% of the cells in the field show staining for GalC, amarker of mature oligodendrocyte lineage cells.

FIG. 8 shows a higher magnification of ES-derived oligodendrocytesstained for GalC (60× magnification). At least ˜10% or 20% of the cellshave morphological characteristics of oligodendrocytes: specifically,numerous processes with webbing in between that is evocative of myelinsheets.

Example 2 hES Derived Oligodendrocytes Cause Remyelination and NeuralSprouting In Vivo

For this example, hES cells form the H7 line were differentiated intooligodendrocyte precursor cells for transplantation using a similarstrategy as in Example 1, with some refinements. The formula for GRM wasthe same, except that the progesterone concentration was recalculated as65 ng/mL. EGF was included in the culture from the outset, and bFGF waswithdrawn after Day 2. TABLE 3 Oligodendroprogenitor Production from hEScells Day: 1 2 3-9 10-15 15-28 28-35 Culture Suspension Add retinoicFull strength Remove RA Remove bFGF Plate on plastic conditions: cultureacid (RA) medium 1 hour; Plate on Matrigel ® overnight Medium: TR TR GRMGRM GRM GRM Additives: bFGF 4 ng/ml bFGF 2 ng/mL EGF 20 ng/mL bFGF 2ng/mL EGF 20 ng/mL EGF 20 ng/mL EGF 20 ng/mL EGF 20 ng/mL RA 10 μM/mLEGF 20 ng/mL Feed every other RA 10 μM/mL day (MWF); non- adherent;complete media replacement

In the first week, feeding was preceded by a 4-5 minute centrifugationat low speed. After spheres start to grow, feeding was done by lettingthe culture sediment for 5-10 minutes in the incubator in 15-50 mLconical tubes. The cells expressing adhesion factors form aggregates(spheres) and sediment faster than the non-adherent cells in single cellsuspension. This increases the purity of cultures with intercellularadhesion characteristics.

FIG. 9 shows the progression of cell morphology during thedifferentiation scheme. (A): Undifferentiated hES cells growing infeeder-free culture on Matrigel® in mEF conditioned medium. (B): By day3, transparent spheres grew from embryoid bodies in suspension culturein medium containing retinoic acid, indicating rapid cell diffusion.(C): Induction of neurolineage cells with retinoic acid (Stage 3)followed by commitment and expansion of oligodendrocyte precursor cellsin the presence of EGF (Stage 4) was evident by the massive accumulationof yellow spheres. (D) Cells were then plated onto Matrigel® forpositive selection of the desired cell type. (E, F): The size of thespheres and percentage of the culture increased as the expansion stageprogressed. After preferential selection, the glial-committedprogenitors could be maintained and amplified for up to 8 weeks. (G, H):Subsequent plating of the oligodendrocyte precursors onto poly-L-lysineand laminin in the absence of mitogens caused them to adopt a morphologyof mature oligodendrocytes within one week.

FIG. 10 shows an immunocytochemical analysis of the matureoligodendrocytes. One week after plating, over 94% of the cells labeledwith antibody to NG2, a marker of early stage glial cells (A). Eightweeks after plating, over 95% of the cells stained positively for GalC(C), O4 (D), and RIP (E). When cells were stained with anti-GFAP andcounterstained with DAPI virtually all of the cells that did not labelwith oligodendroglial markers were GFAP positive (B).

The oligodendrocyte precursor cells were then administered to the lowthoracic spinal cord of anesthetized adult Sprague Dawley rats with amoderate contusion injury of the spine induced using an InfiniteHorizon™ impactor device (Precision Systems and Instrumentation, LLC).

FIG. 11 shows tissue sections obtained after administration of thecells, stained with antibody specific for human nuclear protein. Thedark staining confirms that human cells survive after grafting.

FIG. 12 shows transverse sections of the area around the spinal cord, 9weeks after transplant, stained for human nuclear protein. The animalshad been treated with Cyclosporin A to prevent rejection. The cells havemigrated or proliferated into the white matter.

FIG. 13 shows the results of a detailed analysis to determine whetherthe cell implant has an adverse effect on the site of administration.Serial sections were taken along the length of the spinal cord throughthe injury site of untreated and engrafted animals. Each bar shows theaverage cross-sectional area measured in five sections (mean±SEM).Smaller cross-sectional area near the contusion is the result ofsecondary enlargement following injury. The oligodendrocytes do notinduce sparing, but they also do not make the injury worse. This showsthat hES-derived oligodendrocytes are safe even when administered within1 mm of a spinal cord injury.

FIG. 14 was obtained from an experiment to measure neuronal branching.Eight weeks after injury, animals were injected with BDA as ananterograde tracer of the motor cortex in the corticospinal tract. Thesesections were taken two weeks later. Only in animals treated withhES-derived oligodendrocytes was there evidence of axon branching—shownin the upper two panels as the darkly stained narrow lines. Thebranching was observed in areas of the spinal cord just above theinjury.

FIG. 15 shows quantitation of the neuronal branching measured at 1 mmintervals from the site of injury and cell administration (theepicenter) (mean±SEM for 3 sections per block). Labeled axons werecounted in injured animals that did not receive treatment 3 mm above thesite. Treated animals had labeled axons at a significantly higher levelright up to the epicenter on the rostral side of the injury. Thisconfirms that the engrafted cells are inducing regenerative plasticity.

FIG. 16 is a series of electron micrographs of sections taken fromengrafted animals, showing evidence of substantial remyelination. Thethick circle in the upper left panel is a normally myelinated fiber. Therest of the axons in the field show a thin layer of myelin. The sheathto axon diameter ratio indicates that these axons are newly myelinated.The upper right panel is a higher resolution image of a newly myelinatedaxon. There are about 5 or 6 wraps, and an axonal tongue at the top,representing the leading edge of the oligodendrocyte. This shows thatmyelination of this axon is an ongoing process. The lower panel shows athick myelin sheath being deposited by a Schwann cell. This occursregardless of the transplant, and provides a limited degree of recoveryin untreated animals. However, only the animals engrafted with the hESderived oligodendrocytes showed evidence of myelination of the sortobserved in the upper panels, characteristic of oligodendrocytes.

Example 3 Phenotypic Characterization of Oligodendrocyte Lineage Cells

In this example, marker expression was followed during production ofoligodendrocytes from hES line H7 according to the protocol shown inTable 4. TABLE 4 Oligodendroprogenitor Production from hES cells Day: 12 3-9 10-21 21-28 Culture Suspension Add retinoic acid Full strengthRemove RA Plate on conditions: culture (RA) medium; Matrigel ® RemovebFGF Medium: TR TR GRM GRM GRM Additives: bFGF 4 ng/mL bFGF 2 ng/mL EGF20 ng/mL EGF 20 ng/mL EGF 20 ng/mL EGF 20 ng/mL EGF 20 ng/mL RA 10 μM/mLFeed every other day RA 10 μM/mL (MWF); non-adherent; complete mediareplacement

In the general application of this protocol, oligodendrocyte progenitorssuitable for transplantation are obtained after the solid-phaseselection step (any time after ˜day 23). Culturing in the final stagecan occur to expand the cells as required (at least 8 weeks), bypassaging once a week using trypsin.

For purposes of phenotypic analysis in this example, the cells wereadapted to low-density culture using GRM containing 5% knock-out serumreplacement. The presence of 5% KO-SR starting on day 21 improved theproliferation of bipolar cells when plated on Matrigel®. When the cellswere plated on laminin (15 μg/mL on poly-L-lysine) at low density, theKO-SR improved cell survival and growth. Use of KO-SR earlier in theprotocol did not affect the course of differentiation, as observed bythe growth rate and proportion of yellow spheres.

FIG. 17 shows markers detected on undifferentiated hES cell colonies.Left side panels show in black and white the red fluorescence fromantibody specific for each marker, superimposed on blue fluorescence ofDAPI staining, marking the location of all cell nuclei in the field.Right side panels show the marker staining alone. Comparison of the twoimages allows the viewer to estimate the proportion of total cells inthe field that express the marker in question.

The top row shows colonies positively labeled for SSEA-4, a marker ofpluripotent cells. The bottom row shows stromal cells surrounding thecolonies positively labeled for the mesodermal marker BMP4. Theundifferentiated cells within each colony are BMP4 negative.

FIG. 18 shows the transient appearance of transcription factor Pax6during differentiation (Left side: antibody plus DAPI; right side:antibody staining alone). Top row shows staining towards the center ofthe cluster on day 10, already regulated down in the more differentiatedcells towards the periphery. Bottom row shows virtually no staining incells sampled at day 35.

FIG. 19 shows markers detected in early-stage oligodendrocyte lineagecells, present just after removal of retinoic acid on day 10 (Left side:antibody plus DAPI; right side: antibody staining alone). Top row: thetranscription factor Olig1 (83%±7%). Middle row: the transcriptionfactor SOX10 (72%±12%). Bottom row: non-specific oligodendrocyteprogenitor marker A2B5 (97%±3%).

FIGS. 20A-20B show markers predominating in the fully differentiatedoligoprogenitors at day 35 (Left side: antibody plus DAPI; right side:antibody staining alone). First and second rows (20A): NG2 (chondroitinsulfate proteoglycan, a marker of oligodendrocyte precursors); Third row(20A): GalC; Fourth row (20B): O4; Fifth row (20B): Tuj1 (a marker ofneurons).

The results show that at least about 80% of the cells are positive foroligodendroglial markers NG2, GalC, and O4. Cells that did not labelwith oligodendroglial markers were primarily positive for the neuronmarkers GFAP or Tuj1 (20B, bottom). Double immunocytochemistry showsthat no GFAP or Tuj1 positive cell co-expressed oligodendroglialmarkers. Furthermore, no BMP4 or SSEA-4 could be detected, indicatingthat these cultures were devoid of undifferentiated cells or mesodermallineage cells. This cell population could be caused to proliferatethrough at least eight subsequent passages.

Example 4 hES-Derived Oligodendrocytes Remyelinate Axons inMyelin-Deficient Animals

To demonstrate that the myelination caused by these cells in vivo was adirect effect (rather than by induction of endogenous oligodendrocytes),cells from day 28 of the differentiation protocol were transplanted intothe shiverer mouse model of demyelination. Shiverer mice are homozygousfor a mutation in the myelin basic protein gene located on chromosome 18(Mbpshi/Mbpshi). This gene is duplicated and a large portion of theduplicated gene is inverted, leading to the formation of antisense RNA;this results in severe myelin deficiency throughout the CNS.

Oligodendrocyte progenitor cells were harvested from culture, washed inDMEM, concentrated, and loaded into a silicon coated Hamilton syringe.Animals were immunosuppressed in this as in all experiments with 10mg/kg cyclosporin A. Six weeks later, the animals were sacrificed andsectioned for EM analysis.

FIG. 21 shows the results. Ultrastructurally, axons of shiverer mice aredevoid of myelin or are surrounded by one or two uncompacted wraps ofmyelin (Upper Panel). Six weeks after transplantation of cells, electronmicroscopic analysis indicated multilayered compact myelin indicting themyelinogenic capability of the transplant population (Lower Panel).

Since the mice are deficient in the ability to produce myelin basicprotein, the compact myelin must be produced directly by theadministered oligodendrocytes, and not due to a trophic effect.

Example 5 ES-Derived Oligodendrocytes Restore Spinal Cord Function

Ability of ES-derived oligodendrocyte lineage cells to restoreneurological activity was determined in a rat contusion model of spinalcord injury.

Moderate contusion injury was induced in the low thoracic spinal cord ofanesthetized adult Sprague Dawley rats using an impactor device. Oneweek after the injury, the contusion site was transplanted with 2.5×10⁵cells in 4 μL medium. The transplant cell population was prepared byexposing cells at Day 35 of the differentiation scheme (Example 1) for 3min with trypsin and EDTA to render the cells non-adherent. They werethen rinsed in basal medium, and concentrated to 6×104 cells per μL.

Six weeks after injury, the animals were injected with the anterogradetracer BDA (biotinylated dextran amine) into the motor cortex. Emeraldgreen was injected into the lumbar spine to label lumbar tracts. Thelocation of these markers was determined when the experiment wasterminated at 8 weeks.

FIG. 22 shows the overground locomotion scores for each of the twogroups in this experiment. The upper graph shows the results of animalssubject to a 200 kiloDyne contusion; the lower graph shows results of a250 kiloDyne contusion. The BBB scale is a 21-point scale that ratesnormal overground locomotion based on joint movements, weight support,limb coordination, foot placement and gait stability (Basso, Beattie, &Bresnahan, J. Neurotrauma, 12:1, 1995). (▪) animals treated withES-derived oligodendrocytes 1 week after injury (n=5); (▴) controlanimals receiving the same contusion but no administered cells (n=3).Mean±SEM, for assessments done blinded.

Animals engrafted with hES derived oligodendrocytes showed significantlybetter function (*≡p<0.05), persisting for more than 5 weeks aftertreatment.

These results demonstrate that ES-derived oligodendrocytes help restorefunction in an animal model for spinal cord injury. The earlier examplesshow that transplanted oligodendrocytes cause neuronal sprouting, andremyelination of myelin-deficient axons. Either or both of these effectsmay contribute to the improved behavior observed here.

It is understood that certain adaptations of the invention are a matterof routine optimization, and can be implemented without departing fromthe spirit of the invention, or the scope of the appended claims.

1-34. (canceled)
 35. A differentiated cell population as part of asystem for generating glial cells, wherein at least ˜80% of cells in thedifferentiated cell population are oligodendrocyte precursors having thefollowing characteristics: they are progeny of primate pluripotent stem(pPS) cells; they stain with antibody specific for NG2 proteoglycan; andthey are negative for the neuronal marker NeuN; and wherein the systemfurther comprises the line of pPS cells from which the differentiatedcells were produced.
 36. The differentiated cell population according toclaim 35, wherein at least 80% of the cells also express A2B5.
 37. Thedifferentiated cell population according to claim 35, wherein at least80% of the cells also express platelet-derived growth factor receptor-α(PDGFRα).
 38. The differentiated cell population according to claim 35,wherein at least 20% of the cells show a bipolar morphologycharacteristic of oligodendrocyte precursors.
 39. The differentiatedcell population according to claim 35, wherein culturing of thepopulation for 3 days on poly-L-lysine and laminin in the absence ofmitogens produces cells that express galactocerebroside (GalC).
 40. Thedifferentiated cell population according to claim 35, wherein afterculturing of the population for 3 days on poly-L-lysine and laminin inthe absence of mitogens, at least 10% of the cells have complexprocesses characteristic of mature oligodendrocytes.
 41. Thedifferentiated cell population according to claim 35, whereinimplantation of the population into the spinal cord of a shiverer mutantmouse causes deposition of compact myelin around neuronal axons.
 42. Thedifferentiated cell population according to claim 35, whereinimplantation of the population in or around a contusion injury in thespinal cord of a rat causes improvement in overground locomotion. 43.The differentiated cell population according to claim 35, obtained by aprocess in which the undifferentiated pPS cells are cultured in a mediumcontaining a mitogen and at least two oligodendrocyte differentiationfactors.
 44. The differentiated cell population according to claim 43,obtained by a process in which the undifferentiated pPS cells arecultured in a medium containing fibroblast growth factor (FGF), thyroidhormone, and retinoic acid.
 45. The differentiated cell populationaccording to claim 35, obtained by a process in which glial cells areseparated from non-glial cells.
 46. The differentiated cell populationaccording to claim 35, wherein the pPS cells are human embryonic stem(hES) cells.
 47. A differentiated cell population as part of a systemfor generating glial cells, wherein at least ˜80% of cells in thedifferentiated cell population are oligodendrocyte precursors having thefollowing characteristics: they are progeny of primate pluripotent stem(pPS) cells; they are positive for the transcription factor Olig1; andthey are negative for the neuronal marker NeuN; and wherein the systemfurther comprises the line of pPS cells from which the differentiatedcells were produced.
 48. The differentiated cell population according toclaim 47, wherein at least 80% of the cells also express A2B5.
 49. Thedifferentiated cell population according to claim 47, wherein at least80% of the cells also express platelet-derived growth factor receptor-α(PDGFRα).
 50. The differentiated cell population according to claim 47,wherein at least 20% of the cells show a bipolar morphologycharacteristic of oligodendrocyte precursors.
 51. The differentiatedcell population according to claim 47, wherein culturing of thepopulation for 3 days on poly-L-lysine and laminin in the absence ofmitogens produces cells that express galactocerebroside (GalC).
 52. Thedifferentiated cell population according to claim 47, wherein afterculturing of the population for 3 days on poly-L-lysine and laminin inthe absence of mitogens, at least 10% of the cells have complexprocesses characteristic of mature oligodendrocytes.
 53. Thedifferentiated cell population according to claim 47, whereinimplantation of the population into the spinal cord of a shiverer mutantmouse causes deposition of compact myelin around neuronal axons.
 54. Thedifferentiated cell population according to claim 47, whereinimplantation of the population in or around a contusion injury in thespinal cord of a rat causes improvement in overground locomotion. 55.The differentiated cell population according to claim 47, obtained by aprocess in which the undifferentiated pPS cells are cultured in a mediumcontaining a mitogen and at least two oligodendrocyte differentiationfactors.
 56. The differentiated cell population according to claim 55,obtained by a process in which the undifferentiated pPS cells arecultured in a medium containing fibroblast growth factor (FGF), thyroidhormone, and retinoic acid.
 57. The differentiated cell populationaccording to claim 47, obtained by a process in which glial cells areseparated from non-glial cells.
 58. The differentiated cell populationaccording to claim 47, wherein the pPS cells are human embryonic stem(hES) cells.